This is in the area of the prevention, diagnosis, and treatment of autoimmune diseases having Epstein-Barr virus as an etiological agent.
Epstein-Barr virus infects B cells and induces a large number of different autoantibodies in the early phase of infection. The B cell proliferation and autoantibody production is eventually brought under control in nearly everyone by virus specific T cells. Thereafter, the virus remains latent, surviving in the host for the remainder of the natural life. Once the host is infected the virus continues to “reactivate” at a low level. Evidence for this reactivation is the shedding of virus in the oral cavity, infection through exchange of oral secretions, the spontaneous in vitro outgrowth of transformed B cells, and the spontaneous production of Epstein-Barr virus in vitro. The continuous presence of virus presents a significant challenge to the immune system and requires that the immune mechanisms sustain viral suppression over the many decades of remaining life. If Epstein-Barr virus causes autoimmune disease, then this feature, the sustained presence of a low level of virus in the host continuously emerging from latency, is likely to be important in diseases that appear long after the original infection by Epstein-Barr virus.
Epstein-Barr virus is a herpes virus and is also called Human Herpes Virus 4. This virus is from the genus Lymphocryptovirus and subfamily gammaherpesvirinae. This is the only gamma herpes virus known in man. There are several very good reviews of the biology and structure of Epstein-Barr virus. The reader is referred to classic reviews (Kieff, E. and Liebowitz, D.: Epstein-Barr virus and its replication. In Virology, 2nd ed. Fields et al., eds. pp 1889–1921 (Raven Press, New York 1990); Miller, G.: Epstein-Barr virus. ibid. pp. 1921–1958; Evans, A. S. and Niederman, J. C.: Epstein-Barr virus. In Viral Infections in Humans, 3rd ed. Evans, A. S. ed. pp 265–292 (Plenum, New York City 1989)). Like the other herpes viruses, this is a DNA virus and has a strong propensity for latency. Once latent this virus emerges from latency at a low level throughout life. Epstein-Barr virus induces lymphoma in some non-human primates. In man Epstein-Barr virus appears to be responsible for at least infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma.
Epstein-Barr virus infects the epithelium of the upper airway, B cells and a few T cells. On B cells the viral receptor is the Complement Receptor, Type 2, also known as the CR2 receptor. Infected B cells are able to present antigen, though the virus has recently been found to produce inhibitors of antigen processing (Levitskaya, J. Nature 375:685–688 (1995)), and to synthesize a molecule similar to Il-10 which has profound local effects (Suzuki, T. et al J. Exp. Med. 182:477–486 (1995)). Depending upon what genes are expressed, latently infected B cells may not respond to stimuli in the usual way and may not provide the signals, either qualitatively or quantitatively, that would otherwise be provided. Such aberrant influences upon the normal immune response may provide the basis for subsequent autoimmune disease in some people.
Epstein-Barr virus has been known for more than three decades. For the specific example of an autoimmune disease used herein to illustrate the principles of the invention, many others have considered a relationship between systemic lupus erythematosus and Epstein-Barr virus. The size of the separate literatures concerning lupus, on the one hand, and Epstein-Barr virus on the other are too vast to comprehensively review here. Nevertheless, over 25 years ago antibody titers were noted to be elevated against a number of viruses including rubella, measles, and parainfluenza 1 (Hollinger, F. B. et al. Bact. Proc. 131:174 (1970); Phillips, P. E. and Christian, C. L. Science 168:982–4 (1970); Hurd, E. R. et al. Arthritis Rheum. 13:724–33 (1970)). Perhaps, Dalldorf and colleagues were the first to report an evaluation of the titers of antibody against Epstein-Barr virus in lupus patients; they found no difference between lupus patients and normal controls (Dalldorf, G. et al. J. Am. Med. Assn. 208:1365–8 (1969)).
In contrast, Evans and colleagues claimed to find elevated titers of anti-Epstein-Barr antibodies relative to controls (Evans, A. S., et al. Lancet 1:167–168 (1970)). This paper generated a number of responses, all of which encouraged caution in interpreting these results or address the potential artifacts which could confuse the interpretation (Newell, G. R. and Stevens, D. A. Lancet 1:652 (1971); Evans, A. S. Lancet 1:1023–4 (1971); Gergely, L. et al. Lancet 1:325–326 (1973); Evans, A. S. and Rothfield, N. F. Lancet 1:1127–1128 (1973); Phillips, P. E. et al. Lancet 1:1449 (1973)). Much of the confusion arises from the use of immunofluorescence assays for the detection of anti-Epstein-Barr virus seroconversion. This investigative activity culminated in a remarkable study in which many participants of the controversy combined their resources to develop data they interpreted to show, “the combined approach used in this study fails to provide supportive evidence that E.B. virus is a causative agent in the connective-tissue diseases” (Klippel, J. H. et al. Lancet 2:1057–1058 (1973)). They found no difference in the titer of antibodies against Epstein-Barr virus in lupus compared to controls.
A Japanese group found a high frequency of antibodies against Epstein-Barr virus Nuclear Antigens 2 and 3 in lupus patient sera, compared to normal controls (Kitagawa, H., Et al. Immunol. Lett. 17:249–252 (1988)). Another Japanese group found higher levels of antibody directed against a membrane antigen from Epstein-Barr virus in lupus (and rheumatoid arthritis) sera than in controls (Yokochi, T. et al. J. Rheumatol. 16:1029–1032 (1989)). Similarly, an Australian group found a modest increase in antibodies against early antigens (Sculley, D. G., et al. J. Gen. Virol. 67:2253–2258 (1986)).
An Italian group has shown that the affinity purified antibodies from the 95–119 region of Sm D from lupus patients bind the Epstein-Barr virus Nuclear Antigen-1 between amino acids 35 and 58 (Sabbatini, A., et al. Eur. J. Immunol. 23:1146–1152 (1993)).
The most recent contribution to this question uses both molecular methods to detect Epstein-Barr DNA and serologic methods to detect antibodies (Tsai, Y. et al. Int. Arch. Allergy Immunol. 106:235–240 (1995)). This study also shows no significant differences between lupus patients and controls.
Other diseases, including both rheumatoid arthritis and Sjogren's syndrome, have been explored for a possible relationship to Epstein-Barr virus. Robert Fox and colleagues presented their conception of this area in 1992 (Fox R. I., Luppi, M. and Kang H. J. Rheumatol. 19:18–24 (1992)). The evidence which they conclude supports a role for Epstein-Barr virus in rheumatoid arthritis includes: similarity between synovial and viral antigens, higher levels of antibodies against the Epstein-Barr virus Nuclear Antigens 1 and 3, and the lower ability of lymphocytes to prevent the outgrowth of autologous, Epstein-Barr virus infected lymphocytes (Fox, R. I. Current Opin. Rheum. 7:409–416 (1995)). Others have found a small increase in the frequency of latency for Epstein-Barr virus in rheumatoid arthritis, but a much larger effect for Human Herpes virus-6 (Newkirk, M. M. et al. Br. J. Rhuem. 33:317–322 (1994)).
In Sjogren's syndrome Fox and colleagues note the higher level and frequency of Epstein-Barr virus in salivary gland epithelium and gland tissue (Fox, R. I. et al. J. Immunol 137:3162–3168 (1986)). Other viruses have also been considered by this author (Fox, R. I. Current Opin. Rheum. 7:409–416 (1995)).
Others have developed interesting data from Sjogren's syndrome. Pflugfelder and colleagues found evidence for Epstein-Barr virus in 80% of the lacrimal gland specimens from Sjogren's syndrome patients and in none of the controls (Pflugfelder, S. A. et al Ophthalmology 97:976–984 (1990); and Pflugfelder, S. A. et al. Am. J. Pathol. 143:49–64 (1993)). Karameris and colleagues found higher levels of hybridization between an Epstein-Barr virus DNA probe and the nuclei of salivary gland epithelial cells in Sjogren's syndrome than in controls (Karameris, A. et al. Clin. Exp. Rheum. 10:327–332 (1992)).
Others, however, found no such relationship and concluded that the frequency of Epstein-Barr virus DNA in salivary biopsy specimens was no different in patients with Sjogen's syndrome when compared with normals (Venables, P. J. W., et al. Clin. Exp. Immunol. 75:359–364 (1989); Venables, P. J. W., et al. J. Autoimmunity 2:439–438 (1989); Deacon, L. M., et al. Am J. Med. 92:453–454 (1992)). The data collected by Venables and colleagues were interpreted to show that there was “no evidence that the Epstein-Barr virus infection load is increased. . . [in Sjogren's syndrome]” (Venables, P. J. W. et al. Clin. Exp. Immunol. 75:359–364 (1989)), which is similar to the results of Maitland (Maitland, N. J. Am. J. Med. 96:97 (1994)). Venables and colleagues also refuted there being any abnormality in the serologic response of Sjogren's syndrome patients to Epstein-Barr virus (Deacon, E. M., et al. J. Pathol. 163:351–360 (1991)), citing their data as well as the negative serologic results of Mariette and colleagues (Mariette, X., et al. Am. J. Med. 90:286–294 (1991)).
A Japanese group found an increase in the Epstein-Barr virus production by B cells in patients with Sjogren's syndrome (Tateishi, M. et al. Arthritis Rhuem. 36:827–835 (1993)). Also, Inoue and colleagues found a minor increase in antibody levels against Epstein-Barr virus Nuclear Antigen-2 domains in Sjogren's syndrome compared to controls (Inoue, N. et al. J. Infect. Dis. 164;22–28 (1991)). Another Japanese group reported a modest elevation of anti-Epstein-Barr Nuclear antigen, anti-Early Antigen and anti-Epstein-Barr virus Viral Capsid Antigen (all measured by immunofluorescence) (Toda, I., et al. Sjogren's syndrome (SS) and Epstein-Barr virus (EBV) reactivation. In Lacrimal Gland, Tear Film, and Dry Eye Syndrome. D. A. Sullivan, ed. pp 647–650 (Plenum Press, New York 1994).
Nevertheless, Whittingham has proposed that Epstein-Barr virus is an etiologic agent for Sjogren's syndrome (Whittingham, S., et al. Med. Hypothesis 22:373–386 (1987)). She and her colleagues imagine that the Epstein-Barr viral RNAs called EBER 1 and EBER 2, which are known to bind the La autoantigen, facilitate overcoming tolerance to La and generating autoimmunity. They postulate that the combined effect of Epstein-Barr virus infection and autoimmunity leads to Sjogren's syndrome.
Morshed and colleagues published data showing an increased level of Epstein-Barr virus DNA in patients with primary biliary cirrhosis compared to controls from peripheral blood mononuclear cells, saliva, and fixed liver tissue (Morshed, S. A. et al. Gastroenterol. Jpn. 27:751–758 (1992)). The nuclear dot antigen is an autoantigen bound by autoantibody found in a few sera from patients with primary biliary cirrhosis. This autoantibody is also uncommonly found in lupus and rheumatoid arthritis sera. Analysis of the epitopes of the nuclear dot antigen has revealed two epitopes which have homology with Epstein-Barr virus protein sequences (Xie, K. and Snyder, M. Proc. Natl. Acad. Sci. 92:1639–1643 (1995)).
An example of double infection with Epstein-Barr virus and another virus is found in a cell line isolated from a patient with apparent multiple sclerosis (Haahr, S. et al. Ann. N. Y. Acad. Sci. 724:148–156 (1996)). The increased prevalence of seroconversion among multiple sclerosis patients, relative to controls, has led to the suggestion that Epstein-Barr virus may be an etiologic agent in multiple sclerosis (Sumaya, C. V. et al. Ann. Neurol. 17:371–377 (1985); Bray, P. F., et al. Arch. Neurol. 40:406–408 (1983); Larsen. P. D., et al. Neurology 35:435–438 (1985); Warner, H. B. and Carp. R. I. Med. Hypothesis 25:93–97 (1988); Bray, P. F. et al. Neurology (1992)).
Because of evidence implicating Epstein-Barr virus in infectious mononucleosis, B cell lymphoma (in immunocompromised hosts), burkitt's lymphoma, nasopharyngeal carcinoma, and some cases of Hodgkin's lymphoma, there has been some activity building toward a vaccine against Epstein-Barr virus (Morgan, A. J., et al. J. Med. Virol. 29:74–78 (1989); and Morgan, A. J. Vaccine 10:563–571 (1992)). Recombinant vectors expressing gp340/220 in a bovine papillomavirus vector or in an adenovirus vector protected five of six cottontop tamarins from lymphomas that otherwise occur after infection with Epstein-Barr virus (Finerty, S., et al. J. Gen. Virol. 73:449–453 (1992)). A subunit of the gp340/200 in alum protected three of five cotton top tamarins from lymphomas (Finerty, S., et al. Vaccine 12:1180–1184 (1994)), suggesting that this strategy might not be especially effective. A trial of an Epstein-Barr virus vaccine of gp340/220 in a Vaccinia virus vector has been reported from China and failed to protect a third of those immunized (Gu, S. et al. Dev. Biol. Stand. 84:171–177 (1995)).
A variety of therapies have been attempted against Epstein-Barr virus. These include inducing the lysis cycle in cells latently infected by virus (Gutierrez, M. I., et al. Cancer Res. 56:969–972 (1996)). Patients with the Epstein-Barr virus related lymphomatoid granulomatosis have been treated with interferon-alpha 2b with the preliminary impression that the treatment was successful (Wilson, W. H., et al. Blood 87:4531–4537 (1996)). Cycloheximide has been demonstrated to be useful in vitro (Ishii, H. H., et al. Immunol. Cell Biol. 73:463–468 (1995)). Therapy with a T cell line has been attempted (Kimura, H. et al. Clin. Exp. Immunol. 103:192–298 (1996)), as has adoptive transfer of gene-modified virus-specific T lymphocytes (Heslop, H. E. et al. Nature Med. 2:551–555 (1996)). Data available do not appear to particularly support the use of acyclovir for Epstein-Barr virus infections (Wagstaff, A. J., et al. Drugs 47:153–205 (1994)), though FK506 (a relative of cyclosporine) may have some benefit (Singh, N., et al. Digestive Dis. Sci. 39:15–18 (1994)). Monoclonal antibodies have been used to treat the virus-induced lymphoproliferative syndrome (Lazarovots, A. I., et al. Clin. Invest. Med. 17:621–625 (1994)).
It is therefore an object of the present invention to provide strategies to prevent autoimmune disease by vaccination with vaccines based upon Epstein-Barr virus or upon the structure of Epstein-Barr virus.
It is a further object of this invention to provide vaccines based upon Epstein-Barr virus or upon the structure of Epstein-Barr virus which will have little risk of inducing autoimmune disease.
It is a further object of this invention to provide diagnostics which will identify people exposed to Epstein-Barr virus who are at an increased risk for autoimmune disease and, alternatively, those who are at decreased risk for developing autoimmune disease.
It is a further object of this invention to provide for the application of antiviral therapy directed against Epstein-Barr virus in the treatment of autoimmune disease.
It is a further object of this invention to provide diagnostics and therapeutics for autoimmune disease based upon the changes induced by the host by Epstein-Barr virus.